Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Measure of STAT6 mRNA level in HT-29 cells 24 hours post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (B) STAT6 protein level analysis at 2, 5 and 7 days post-transfection in HT-29 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Measure of STAT6 mRNA level in ZR-75-1 cells 3 days post-transfection. The graph represents the mean ± SEM of multiple independent experiments (n) obtained by real-time PCR. Results were analysed by ΔΔCt method for relative quantifications. The fold change is represented by the Y axis, and values are normalized to control cells. (D) STAT6 protein level analysis after 4 and 7 days of transfection in ZR-75-1 cells. The graph represents the mean of the percentage of STAT6 positive cells ± SEM of multiple independent experiments (n) obtained by flow cytometry. E) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells (top) and ZR-75-1 (bottom) cells. STAT6 siRNA sequences and non-targeting siRNA were used at 100 nM as the final concentration. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Concentration Assay

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A and B) Number of live HT-29 cells measured at 5 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (C) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent experiments (n) shown in A and B. (D and E) Number of live ZR-75-1 cells measured at 4 and 7 days post-transfection, respectively. The graphs represent the mean ± SEM of multiple independent experiments (n). (F) The graph illustrates how ZR-75-1 cells grew over time and represents the mean ± SEM of the multiple independent experiments (n) shown in D and E. The number of live cells was calculated as detailed in the material and methods using NucleoCounter NC-100. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the transfection with STAT6 siRNA sequences. STAT6 and non-targeting (NT) siRNA sequences were used at 100 nM as the final concentration. Non-transfected cells served as negative control and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Comparison, Concentration Assay, Negative Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) Apoptosis analysis in HT-29 cells. (B) Apoptosis analysis in ZR-75-1 cells. In both cases, the graphs represent from left to right: early (Annexin V + /PI - ), late (Annexin V + /PI + ) and total (Annexin V + ) apoptosis. The graphs represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. (C) Representative flow cytometry plots in HT-29 cells. (D) Representative flow cytometry plots in ZR-75-1 cells. The X axes represent Annexin V and the Y axes represent PI fluorescence intensity. Quadrants were set according to cells independently stained with Annexin V or PI. Apoptosis was studied at 7 days post-transfection in both cell lines. Data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Flow Cytometry, Fluorescence, Staining, Transfection, Software, Sequencing, Concentration Assay, Control

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: STAT6 siRNA transfection was carried out at day 1 of cell culture with (A) STAT6.1 and (B) STAT6.4 at 100 nM. A second transfection was carried out in both cases with STAT6.1 and STAT6.4 at the same concentration 7 days after the first transfection. The graphs represent the number of live cells over time measured at day 7 and 14 days post-first transfection counted using NucleoCounter NC-100 as detailed in the material and methods section. The mean ± SEM of 3 independent experiments is represented in each graph. Control cells were non-transfected cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The percentage of reduction of the number of live cells was calculated by comparison between the mean of NT vs . the mean of the individual transfection with STAT6 siRNA sequences, and sequential transfection with NT (NT+NT) vs . sequential transfection with STAT6.1 and STAT6.4.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Transfection, Cell Culture, Concentration Assay, Control, Comparison

Journal: PLoS ONE

Article Title: STAT6 knockdown using multiple siRNA sequences inhibits proliferation and induces apoptosis of human colorectal and breast cancer cell lines

doi: 10.1371/journal.pone.0207558

Figure Lengend Snippet: (A) STAT6 expression at protein level in HT-29 cells at day 2 and 7 post-transfection measured by flow cytometry. The graph represents the mean ± SEM of multiple independent experiments (n). Data was analysed using Flowjo Software. The percentage of STAT6 positive cells is represented on the Y axis. (B) Representative histograms (Control: back; NT: grey; STAT6.1: red; STAT6.4: blue) of STAT6 protein analysis at 7 days post-transfection by flow cytometry in HT-29 cells. (C) Number of live HT-29 cells measured at day 7 post-transfection by NucleoCounter NC100. The graph represents the mean ± SEM of multiple independent experiments (n). (D) The graph illustrates how HT-29 cells grew over time and represents the mean ± SEM of the independent number of experiments shown in C. (E, F and G) The graphs show early (E) (Annexin V + /PI - ), late (F) (Annexin V + /PI + ) and total (G) (Annexin V + ) apoptosis, and represent the mean ± SEM of multiple independent experiments (n) obtained by flow cytometry. Apoptosis was studied at 7 days post-transfection. Data were analysed with Flowjo Software. STAT6 siRNA sequences and a non-targeting siRNA sequence were used at 100 nM as the final concentration. Non-transfected cells served as control cells and STAT6 siRNA sequences 1 and 4 and non-targeting siRNA are denoted as STAT6.1, STAT6.4 and NT, respectively. The number of independent experiments (n) is set out in the Fig.

Article Snippet: Cells were washed twice with PBS/0.5%BSA (Bovine Serum Albumin) and stained with anti-STAT6 APC conjugated antibody (Miltenyi Biotec, 130-104-030) (20 μl/10 6 cells) for 30 min in the dark at 4°C.

Techniques: Expressing, Transfection, Flow Cytometry, Software, Control, Sequencing, Concentration Assay

Journal: Cell proliferation

Article Title: Tetrahedral Framework Nucleic Acid Relieves Sepsis-Induced Intestinal Injury by Regulating M2 Macrophages.

doi: 10.1111/cpr.13803

Figure Lengend Snippet: FIGURE 4 | Treatment of tFNAs activated the Mertk signal pathway. (A) Representative images of immunofluorescent staining for tFNAs (red) and DAPI (blue) in the colon and ileum (Scale bar = 20 μm). (B) Immunohistochemical staining of Mertk in colon (scale bar: 100 μm). (C–F) Western blotting detection of Mertk, p-Mertk, STAT1, p-STAT1, SOCS1, SOCS3 expression in the colon. (G) Quantitative analysis of the protein expression levels. (H) Schematic diagram of the inhibition of Mertk altering macrophage activation by tFNAs. (I–L) Western blotting detection of Mertk, p- Mertk, STAT1, p-STAT1, SOCS1, SOCS3 expression in the colon. (M) Quantitative analysis of the protein expression levels. Data were represented as mean ± SD, *p < 0.05, **p < 0.01.

Article Snippet: The antibody for ZO- 1 was from Wuhan Servicebio; the antibody for occludin was from Abcam; the antibodies for Mer, STAT1, p- STAT1, SOCS1 and SOCS3 were from Cell Signalling Technology; the antibody for p- Mer (Tyr749) was from Thermo Fisher Scientific; and UNC2250 was purchased from MCE.

Techniques: Staining, Immunohistochemical staining, Western Blot, Expressing, Inhibition, Activation Assay

Journal: BMC cancer

Article Title: NR1D1 suppressed the growth of ovarian cancer by abrogating the JAK/STAT3 signaling pathway.

doi: 10.1186/s12885-021-08597-8

Figure Lengend Snippet: Fig. 2 NR1D1 induced cell cycle arrest and apoptosis in ovarian cancer cells. A Cell cycle of NR1D1 over-expressed cells was determined by flow cytometry. B The levels of cyclinD and cyclinE were determined by western blot. C Flow cytometry was performed to determine the cell cycle of NR1D1 silenced cells. D Western blot was performed to determine the levels of cyclinD and cyclinE. E Apoptosis of NR1D1 over-expressed cells was determined by flow cytometry. F After transfection with NR1D1 over-expression plasmid, the levels of activated caspase-3 and caspase-9 were determined. The results are presented as mean + SD

Article Snippet: Following blocking with 5% bovine serum albumin, the membranes were incubated with antibodies against NR1D1 (1:1000; Abclonal, Wuhan, China), cyclinD (1:1000; ABclonal), cyclinE (1:1000; Proteintech, Wuhan, China), SOCS3 (1:1000; ABclonal), JAK-1 (1:1000; Affinity, Changzhou, China), p-JAK1 (Tyr 1034/Tyr 1035; 1:1000; Affinity), JAK2 (1:500; Affinity), p-JAK2 (Tyr 1007/Tyr 1008, 1:1000; Affinity), STAT3 (1:500; Affinity), p-STAT3 (Tyr 705, 1:500; Affinity), β-actin (1:2000; Proteintech) at 4 °C overnight.

Techniques: Flow Cytometry, Western Blot, Transfection, Over Expression, Plasmid Preparation

Journal: Viruses

Article Title: ZIKV Strains Elicit Different Inflammatory and Anti-Viral Responses in Microglia Cells

doi: 10.3390/v15061250

Figure Lengend Snippet: Infection of BV2 microglia cells with ZIKV PE243 and ZIKV MR766 strains differentially modulate the pro-inflammatory miRNA-155 and anti-inflammatory and anti-viral factors. BV2 cells were infected with both ZIKV PE243 or ZIKV MR766 , with an MOI of 1, at 12, 24, 48, and 72 hpi using mock-treated (Mock) as a negative control and LPS + ATP (LPS) as a positive control. ( a ) miRNA-155, ( b ) SOCS1, ( d ) SOCS3 and ( e ) SHIP1 mRNA expression levels were measured by RT-qPCR. ∆∆Ct plotted results were normalized to hprt1 and mock values for mRNAs, and U6 and mock for miRNAs. ( c ) The protein SOCS1 expressions were analyzed by Western analysis. The membrane was stained with anti-SOCS1 and anti-β-actin as a loading control. Statistical analysis was performed by One-Way ANOVA, considering * p < 0.05, ** p < 0.01, and *** p < 0.001 as significant differences.

Article Snippet: Primary antibodies against PPAR-γ (Thermo Fisher Scientific Inc., PA3-821A) and SOCS1 (Boster Biological Technology Co. Ltd, Pleasanton, CA, USA, PA1074) were added to the membranes, and then secondary antibodies against mouse (Cell Signaling Technology, 7076S, Danvers, MA, USA) and rabbit (KPL, 04-15-06) were used.

Techniques: Infection, Negative Control, Positive Control, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Staining, Control

Journal: Viruses

Article Title: ZIKV Strains Elicit Different Inflammatory and Anti-Viral Responses in Microglia Cells

doi: 10.3390/v15061250

Figure Lengend Snippet: Comparison of inflammatory, anti-inflammatory, and anti-viral marker expressions by BV2 microglial cell line after infection with Brazilian and African ZIKV strains (ZIKV PE243 and ZIKV MR766 ).

Article Snippet: Primary antibodies against PPAR-γ (Thermo Fisher Scientific Inc., PA3-821A) and SOCS1 (Boster Biological Technology Co. Ltd, Pleasanton, CA, USA, PA1074) were added to the membranes, and then secondary antibodies against mouse (Cell Signaling Technology, 7076S, Danvers, MA, USA) and rabbit (KPL, 04-15-06) were used.

Techniques: Comparison, Marker, Infection

Journal: BMC genomics

Article Title: Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation.

doi: 10.1186/1471-2164-13-731

Figure Lengend Snippet: Figure 9 Expression of HK2 and STAT5 mRNA in the bovine mammary gland. Expression levels of HK2 and STAT5 in lactating and non-lactating bovine mammary glands were determined using real-time PCR. Gene fold changes were calculated using the 2−ΔCt

Article Snippet: Antibodies for STAT5 and β-actin were manufactured by Boster (China), and the HK2 antibody was purchased from Santa Cruz (USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC genomics

Article Title: Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation.

doi: 10.1186/1471-2164-13-731

Figure Lengend Snippet: Figure 8 Putative miRNAs targeting gene 3’UTRs. (A) Pairing of bta-miR-141 with the bovine STAT5 3’UTR sequence. (B) A target seed region of the miRNA binding site was found within the bovine HK2 3’UTR sequence.

Article Snippet: Antibodies for STAT5 and β-actin were manufactured by Boster (China), and the HK2 antibody was purchased from Santa Cruz (USA).

Techniques: Sequencing, Binding Assay

Journal: BMC genomics

Article Title: Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation.

doi: 10.1186/1471-2164-13-731

Figure Lengend Snippet: Figure 10 STAT5 protein is regulated by miR-141. STAT5 and β-actin protein expression was determined by western blotting. Mac-T cells were transfected with an miR-141 inhibitor, an miR-141 mimic, an inhibitor negative control (INC) and a negative control (NC). Cell lysates were collected after 24 hours and analyzed by western blotting. Beta-actin served as the internal control. The protein fragment intensity was quantified using Imagpro-Plus software, and IOD was used to represent the protein level. Error bars represent SD. n=3. a-c means different superscripts differ (P<0.05).

Article Snippet: Antibodies for STAT5 and β-actin were manufactured by Boster (China), and the HK2 antibody was purchased from Santa Cruz (USA).

Techniques: Expressing, Western Blot, Transfection, Negative Control, Control, Software